Microorganism Producing Inosine and Method of Producing Inosine Using the Same

ABSTRACT

The present invention relates to a microorganism producing inosine, which is one of purine nucleoside, an important material for 5′-inosinic acid synthesis, and method for producing inosine using the same. More particularly, the present invention relates to a recombinant microorganism of  Corynebacterium  genus producing inosine at high concentration by inactivating the gene encoding nucleoside hydrolase II and by enhancing the expression of the gene encoding 5′-nucleotidase, which still retains the characteristics of  Corynebacterium ammoniagenes  CJIP2401 (KCCM-10610).

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/522,004, filed on Jul. 2, 2009, which is a 35 U.S.C. §371 nationalphase application of PCT/KR2008/000236 (WO 2008/088156), filed on Jan.15, 2008, which claims the benefit of Korean patent application serialno. KR 10-2007-0004341, filed on Jan. 15, 2007. Each of theseapplications is specifically incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to a microorganism producing inosine, oneof purine nucleosides which are very important materials for 5′-inosonicacid synthesis, and method for producing inosine using the same. Moreparticularly, the present invention relates to a recombinantmicroorganism of Corynebacterium genus producing inosine at highconcentration, in which the characteristics of Corynebacteriumammoniagenes CJIP2401 (KCCM-10610) are exhibited, the gene encodingnucleoside hydrolase II (referred as ‘rih II’ hereinafter) isinactivated and expression of the gene encoding 5′-nucleotidase(referred as ‘ushA’ hereinafter) is enhanced, and method for producinginosine using the same.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “sequence listing.txt”, created Sep. 7, 2011, size of 6kilobytes.

BACKGROUND ART

As a nucleoside synthesis-related enzyme, nucleoside hydrolase II isknown to be involved in the degradation of nucleoside and5′-nucleotidase is known to be involved in the nucleoside synthesis.

A gene encoding Nucleoside hydrolase II is the gene encoding the enzymecatalyzing ribose and corresponding bases, which are purine andpyrimidine, irreversibly, in salvage pathway of nucleoside. This genecontains a 1326 by nucleoside sequence and encodes a protein of 308amino acids and has substrate-specificity to both of purine andpyrimidine nucleosides (Microbiology, 2006, vol. 152, p 1169-1177).

A gene encoding 5′-nucleotidase is the gene encoding the enzymecatalyzing dephosphorylation of nucloeside such as AMP, GMP, IMP and XMPin nucleoside biosynthesis pathway (de novo pathway). This gene encodesa protein of 705 amino acids.

Inosine is an important substrate for the chemical synthesis andsynthesis by enzyme transfer of 5′-inosinic acid which is in the limelight as a flavor-enhancing seasoning. Also, inosine is an intermediateof nucleic acid biosynthesis, which is not only an importantphysiological factor in animals and plants but also applied in variousfields including food and medicinal industries. Nucleoside and itsderivatives have been reported to have many usages as antibiotic,anti-cancer and anti-viral substances (J. Virol, vol; 72, pp 4858-4865).

The conventional method to produce inosine is direct fermentation usingsuch microorganism as Bacillus (Agric. Biol. Chem., 46, 2347 (1982);Korean Patent No. 27280) or Corynebacterium ammoniagenes (Agric. Biol.Chem., 42, 399(1978)), or thermal decomposition of 5′-inosinic acid(Japanese Laid-off Patent Publication No. S43-3320). However, thethermal decomposition requires massive heat, which makes it notpractical. According to the direct fermentation, activity of the strainproducing inosine is very low, so that production cost increases andfermentation takes longer, resulting in the low productivity.

To produce inosine by direct fermentation using a microorganism at highconcentration with high yield, it is very important to develop a strainthat is appropriate for the accumulation of inosine at highconcentration in its culture broth. The present inventors studiedpersistently to produce inosine with high productivity/high yield bydirect fermentation using a microorganism. And at last, the presentinventors completed this invention by confirming that the microorganismproducing 5-inosonic acid could produce inosine with highproductivity/high yield when rih II gene encoding nucleoside hydrolaseII is inactivated and ushA gene encoding 5′-nucleotidase is enhanced.

DISCLOSURE OF THE INVENTION

The present invention is established based on the above finding.Therefore, it is an object of the present invention to provide arecombinant microorganism of Corynebacterium genus wherein rih II geneencoding nucleoside hydrolase II is inactivated and the expression ofushA gene encoding 5′-nucleotidase is enhanced.

It is another object of the present invention to provide a method forproducing inosine using the above microorganism.

The above objects and other objects of the present invention can beachieved by the following embodiments of the present invention.

The present invention is described in detail hereinafter.

To achieve the objects of the invention, the present invention providesa microorganism having nucleoside productivity by manipulating the genesinvolved in nucleoside synthesis pathway of CJIP2401 (KCCM-10610), themutant strain of Corynebacterium ammoniagenes ATCC 6872 that is able toaccumulate 5′-inosinic acid at high concentration.

The parent strain used in the present invention, Corynebacteriumammoniagenes CJIP2401 (KCCM-10610) [Korean Patent Publication No.10-2004-0099466], is the mutant strain of Corynebacterium ammoniagenesMP377 (KFCC11141). The mutant strain of Corynebacterium ammoniagenesMP377 (KFCC11141) [Korean Patent Application No. 10-2000-0013303] isoriginated from Corynebacterium ammoniagenes KFCC-10814 [Korean PatentNo. 127853], the mutant strain of Corynebacterium ammoniagenes 6872.Therefore, the microorganism of the present invention shares the majorcharacteristics explained below and has similar effects withCorynebacterium ammoniagenes CJIP2401 (KCCM-10610).

1) Adenine auxotroph

2) Guanine leaky type

3) Biotin auxotroph

4) sensitivity to Lysozyme 8 g/ml

5) resistant to 3,4-dihydroproline 3500 g/ml

6) resistant to 6-mercaptopurine 300 g/ml

7) resistant to 5-fluorotryptophan 10 mg/l

In this invention, rih II gene encoding the nucleoside hydrolase II wasisolated from the wild type Corynebacterium ammoniagenes ATCC 6872 andpreferably had the sequence represented by SEQ. ID. NO: 1.

In this invention, ushA gene encoding 5′-nucleotidase was isolated fromthe wild type Corynebacterium ammoniagenes ATCC 6872 and preferably hadthe sequence represented by SEQ. ID. NO: 2.

The present invention provides recombinant microorganisms of theCorynebacterium genus in which rih II gene encoding nucleoside hydrolaseII protein is inactivated.

The present invention also provides a recombinant microorganism ofCorynebacterium genus in which rih II gene encoding nucleoside hydrolaseII protein is inactivated and the expression of ushA gene encoding5′-nucleotidase is enhanced.

In this invention, the “inactivation” can be induced by any inactivationmethod known to those in the art. The term “inactivation” herein intendsto mean that the expression of rih II gene encoding the nucleosidehydrolase II is reduced to a low level compared to the wild type strain,or genes that are not expressed and genes that express products havingno activity or reduced activity in spite of being expressed areproduced.

In this invention, the “inactivation” can be induced by one or moremutation methods selected from the group consisting of insertion of oneor more base pairs in rih II gene, deletion of one or more base pairs inthe gene, transition or transversion of base pairs by inserting nonsensecodon in the gene.

In a preferred embodiment of the present invention, the microorganismcontaining the inactivated gene can be obtained by culturing amicroorganism of Corynebacterium genus transformed with the vectorcontaining a part of the rih II gene.

The part of the gene can be synthesized by using primers represented bySEQ. ID. NO: 3 and NO: 4.

In this invention, the rih II gene was inactivated by the insertion of arecombinant vector into Corynebacteria. However, instead of therecombinant vector, any known method including virus infection can beused to inactivate rih II gene, but not always limited thereto.

In this invention, the enhancement of the gene expression can beperformed by any conventional method known to those in the art. Herein,the reinforcement of gene expression means the up-regulation of ushAgene encoding 5′-nucleotidase, compared with the wild type strain.

In this invention, the enhancement of the gene expression is induced byactivation of the entire expression system containing the promoterregion of ushA gene encoding 5′-nucleotidase.

In a preferred embodiment of the present invention, the microorganismcontaining the enhanced gene can be obtained by culturing amicroorganism of Corynebacterium genus transformed with the vectorcontaining the sequence represented by SEQ. ID. NO: 2 to express theentire expression system including the promoter region of ushA geneencoding 5′-nucleotidase.

The present invention further provides Corynebacterium ammoniagenesCJIG650 (Accession No: KCCM-10828P) having inactivated rih II gene andproducing 5′-inosinic acid and inosine at high concentration, which isthe mutant strain of Corynebacterium ammoniagenes CJIP2401 (KCCM10610).

The present invention also provides Corynebacterium ammoniagenes CJIG651(Accession No: KCCM-10829P) having inactivated rih II gene and enhancedushA gene and producing inosine at high concentration, which is themutant strain of Corynebacterium ammoniagenes CJIG650 (KCCM-10828P).

The present invention also provides a method for producing inosine athigh concentration with high yield by culturing Corynebacteriumammoniagenes CJIG650 (KCCM-10828P) and CJIG651 (KCCM-10829P) andaccumulating inosine in the culture broth.

The construction process of the strain of the present invention is asfollows.

Sequencing was performed with chromosome of a microorganism ofCorynebacterium genus producing 5′-inosinic acid for exampleCorynebacterium ammoniagenes ATCC 6872. As a result, it was confirmedthat the size of rih II gene encoding nucleoside hydrolase II wasapproximately 1326 by (SEQ. ID. NO: 1). PCR was performed to obtain thegene fragment to construct a recombinant vector. The microorganism ofCorynebacterium genus producing 5′-inosinic acid was transformed withthe vector and the strain producing 5′-inosinic acid and inosine at highconcentration was selected from the transformed strains. From thesequencing with chromosome of Corynebacterium ammoniagenes ATCC 6872, itwas confirmed that the size of ushA gene encoding 5′-nucleotidase wasapproximately 2115 by (SEQ. ID. NO: 2). PCR was performed to obtain thegene fragment to construct a recombinant vector. The microorganism ofCorynebacterium genus producing 5′-inosinic acid and inosine wastransformed with the vector and the strain producing inosine alone athigh concentration was selected from the transformed strains.

The microorganism of the present invention is preferably obtained bytransforming the microorganism of Corynebacterium genus producing5′-inosinic acid with the recombinant vector containing rih II geneencoding nucleoside hydrolase II and the recombinant vector containingthe ushA gene encoding 5′-nucleotidase. The recombinant vectorcontaining nucleoside hydrolase II gene includes 500 by of rih IIstructural gene sequence, while the recombinant vector containing5-nucleotidase gene includes approximately 300 by of promoter, ushAstructural gene sequence and approximately 300 by of terminator. Themicroorganism of the present invention is generated by disrupting thegene by inserting the recombinant vector containing rih II geneconstructed using chromosome of the microorganism of Corynebacteriumgenus producing 5′-inosonic acid and by over-expressing ushA gene as aform of plasmid.

The recombinant vector containing nucleoside hydrolase II gene isconstructed by the following processes; rih II gene obtained by PCR isseparated and purified; and the purified gene is inserted intopCR2.1-TOPO vector by ligation. The recombinant vector herein ispreferably pTOPO-rih II vector constructed by inserting a part ofstructural gene of nucleoside hydrolase II into pCR2.1-TOPO vector. Therecombinant vector containing 5′-nucleotidase gene is constructed by thefollowing processes; ushA gene obtained by PCR is digested with EcoRVand BamHI; and the gene fragment is inserted into the vector predigestedwith the same DNA restriction enzymes by using DNA T4 ligase. Theapplicable vector is not limited to specific one, and any conventionalvector informed to those in the art can be used. However, pECCG117vector [Biotechnology letters vol 13, No. 10, p. 721-726(1991) or KoreanPatent Publication No. 92-7401] is preferred and particularlypECCG117-ushA vector constructed by inserting ushA gene into peCCG117vector is preferred.

Using the recombinant vector, for example pTOPO-rih II, linear DNAfragment can be inserted into the microorganism (for example,Corynebacterium ammoniagenes CJIP2401 (KCCM-10610)) by generalelectroporation and the microorganism is cultured in the mediumcontaining kanamycin, the antibiotic used as a selection marker,followed by selection. The insertion of pTOPO-rih II vector in theselected transformed strain can be confirmed by PCR.

To over-express ushA gene in the selected transformed strain, linear DNAfragment is inserted into the selected transformed strain usingpECCG117-ushA vector by electroporation and the strain is cultured inthe medium containing chloramphenicol, the antibiotic used as aselection marker, followed by selection. The insertion of pECCG117-ushAvector in the selected transformed strain can be confirmed by PCR.

The microorganism of the present invention can be cultured in theconventional medium containing proper carbon source, nitrogen source,amino acids and vitamins, under aerobic condition with regulatingtemperature and pH properly.

As a carbon source, one of carbohydrates selected from the groupconsisting of glucose, fructose, and sterilized pretreated molasses(molasses converted to reducing sugar). The nitrogen source isexemplified by such inorganic nitrogen source as ammonia, ammoniumchloride and ammonium sulfate and such organic nitrogen source aspeptone, NZ-amine, gravy, yeast extract, corn steep liquor, caseinhydrolysate, fishes or decomposition product thereof, and defattedsoybean cake or degradation products thereof. The medium herein canadditionally include such inorganic compounds as potassium dihydrogenphosphate, dipotassium hydrogen phosphate, magnesium sulfate, ironsulfate, manganese sulfate and calcium sulfate, etc. In addition,vitamins and auxotrophic bases can be added as well. The culture isperformed under aerobic condition by shaking-culture or submergerculture preferably at 20˜40° C. pH is preferably regulated aroundneutral over the culture. The duration of the culture is preferably 4˜5days. The inosine accumulated by direct fermentation can be recovered bythe conventional method.

According to the method of the present invention, inosine can beproduced with high yield.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a diagram illustrating the process of cloning of rih II genefragment encoding nucleoside hydrolase II originated from the wild typeCorynebacterium ammoniagenes ATCC 6872, and the cloned vector pTOPO-rihII.

FIG. 2 is a diagram illustrating the process of cloning of ushA genefragment encoding 5′-nucleotidase originated from the wild typeCorynebacterium ammoniagenes ATCC 6872, and the cloned vectorpECCG117-ushA.

BEST MODE FOR CARRYING OUT THE INVENTION

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

EXAMPLE 1 Cloning of rih II Gene

In this example, 1326 by rih II gene fragment (SEQ. ID. NO: 1) wasamplified by PCR using chromosomal DNA of the wild type Corynebacteriumammoniagenes ATCC 6872 as a template with primers of oligonucleosidesrepresented by SEQ. ID. NO: 3 and NO: 4 to construct the vector (rih IIgene disruption vector) containing a part of rih II gene encodingnucleoside hydrolase II and an antibiotic marker. PCR was performed asfollows; denaturation at 96° C. for 30 seconds, annealing at 52° C. for30 seconds, and polymerization at 72° C. for 30 seconds (30 cycles). Theamplified rih II gene fragment was cloned into the plasmid pCR2.1-TOPO(Invitrogen Co., USA). As a result, pTOPO-rih II vector was obtained.The cloning process of rih II gene fragment and the cloned vectorpTOPO-rih II are illustrated in FIG. 1.

Corynebacterium ammoniagenes CJIP2401 (KCCM-10610) was transformed withthe constructed pTOPO-rih II vector by electroporation. Then, singlecolonies growing on CM solid medium (gravy 10 g/L, yeast extract 10 g/L,bactopeptone 10 g/L, sodium chloride 2.5 g/L, bactoagar 1.7%, pH 7.0)containing kanamycin (25 mg/L) were recovered.

The colonies were cultured in CM medium containing the same antibiotic,followed by separation of plasmid. The size of the plasmid was measuredfirst and then colony PCR was performed using the primers comprisingboth ends of multi-cloning region of pCR2.1-TOPO to confirmtransformation by measuring the size of the inserted DNA.

Primers used for inactivating rih II gene are as follows.

Primer rih II-F (SEQ. ID. NO: 3): 5′-TGCTGGCGATGCACTTGAAT-3Primer rih II-B (SEQ. ID. NO: 4): 5′-TGTGCGACCAATGGTGGGGCC-3′

The strain containing rih II gene selected above was namedCorynebacterium ammoniagenes CJIG650, which was deposited at KCCM(Korean Culture Center of Microorganisms) of KFCC (Korean Federation ofCulture Collection) on Dec. 15, 2006 (Accession No: KCCM 10828P).

EXAMPLE 2 Cloning of ushA Gene

In this example, 2115 by ushA gene fragment (SEQ. ID. NO: 2) wasamplified by PCR using chromosomal DNA of the wild type Corynebacteriumammoniagenes ATCC 6872 as a template with primers of oligonucleosidesrepresented by SEQ. ID. NO: 5 and NO: 6. PCR was performed as follows;denaturation at 96° C. for 30 seconds, annealing at 52° C. for 30seconds, and polymerization at 72° C. for 30 seconds (30 cycles). Theobtained gene fragment was digested with EcoRV and BamHI. The genefragment was ligated to linear pECCG117 vector digested with the sameDNA restriction enzymes by using DNA T4 ligase. As a result,pECCG117-ushA vector was obtained. The cloning process of ushA genefragment and the cloned vector pECCG117-ushA are illustrated in FIG. 2.

The mutant strain prepared in Example 1 was transformed with theconstructed pECCG117-ushA vector by electroporation. Then, singlecolonies growing on CM solid medium (gravy 10 g/L, yeast extract 10 g/L,bactopeptone 10 g/L, sodium chloride 2.5 g/L, bactoagar 1.7%, pH 7.0)containing chloramphenicol (7.5 mg/L) were recovered. The colonies werecultured in CM medium containing the same antibiotic, followed byseparation of plasmid. The size of the plasmid was measured first andthen colony PCR was performed using the primers comprising both ends ofmulti-cloning region of pECCG117 to confirm transformation by measuringthe size of the inserted DNA.

Primers used for over-expressing ushA gene are as follows.

Primer ushA-F (SEQ. ID. NO: 5): 5′-GTGTCTAAGTTTCGCCGTTTTGGC-3Primer ushA-B (SEQ. ID. NO: 6): 5′-GCCGGATCCCTAGAATTTGATGTGGCTAACCTCG-3′

The strain containing ushA gene selected above was named Corynebacteriumammoniagenes CJIG651, which was deposited at KCCM (Korean Culture Centerof Microorganisms) of KFCC (Korean Federation of Culture Collection) onDec. 15, 2006 (Accession No: KCCM 10829P).

EXAMPLE 3 Fermentation Test In Erlenmeyer Flask

3 ml of seed medium was distributed in test tubes of 18 mm in diameter,which were sterilized by autoclave. Corynebacterium ammoniagenes CJIG650(KCCM-10828P), CJIG651 (KCCM-10829P) and the parent strain (KCCM-10610)were inoculated in the test tubes respectively, followed by shakingculture at 30° C. for 24 hours to prepare seed culture solution. 27 mlof fermentation medium was distributed in 500 ml Erlenmeyer flask forshaking, which was sterilized by autoclave at 120° C. for 10 minutes.After inoculation of 3 ml of the seed culture solution, culture wasperformed for 4˜5 days. RPM was regulated as 200 and temperature was setat 32° C. and pH was regulated as 7.2.

The compositions of the seed medium and the fermentation medium are asfollows.

Seed medium: Glucose 5%, Peptone 0.5%, Gravy 0.5%, Yeast extract 1%,Sodium chloride 0.25%, Adenine 100 mg/l, Guanine 100 mg/l, pH7.2

Flask fermentation medium: Sodium glutamate 0.1%, Ammonium chloride 1%,Magnesium sulfate 1.2%, Calcium chloride 0.01%, Iron sulfate 20 mg/l,Manganese sulfate 20 mg/l, Zinc sulfate 20 mg/l, Copper sulfate 5 mg/l,L-cysteine 23 mg/l, Alanine 24 mg/l, Nicotinic acid 8 mg/l, Biotin 45μg/l, Thiamine HCl 5 mg/l, Adenine 30 mg/l, Phosphoric acid (85%) 1.9%,Reducing sugar 8% converted from the mixture of fructose, glucose andmolasses.

The accumulations of inosinic acid (IMP) or inosine in CJIG650(KCCM-10828P), CJIG651 (KCCM-10829P) and the parent strain (KCCM-10610)culture media were compared one another. And the results are shown inTable 1. As shown in Table 1, only inosinic acid was accumulated in theparent strain KCCM-10610) culture medium and inosine was not accumulatedat all therein. However, the strains of the present invention, CJIG650(KCCM-10828P) produced inosinic acid and inosine at high concentration,and the strain CJIG651 (KCCM-10829P) produced inosine at highconcentration.

Therefore, the strains of the present invention CJIG650 (KCCM-10828P)and CJIG651 (KCCM-10829P) were confirmed to be the strains capable ofproducing inosine at high concentration.

TABLE 1 Strain IMP concentration (g/L) Inosine concentration (g/L)Control 14.1 — (KCCM-10610) CJIG650 8 4 CJIG651 — 8.3

INDUSTRIAL APPLICABILITY

As explained hereinbefore, the Corynebacterium ammoniagenes CJIG650 andCJIG651 prepared by inactivating the gene encoding nucleoside hydrolaseII and by reinforcing the gene encoding 5′-nucleotidase can produceinosine at high concentration

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A method for producing inosine by fermentation comprising: culturinga microorganism belonging to Corynebacterium ammoniagene in a culturemedium to produce and accumulate inosine in the medium, and collectinginosine, wherein, said microorganism has inosine-producing ability andis modified to inactivate the gene encoding nucleoside hydrolase IIhaving the nucleotide sequence represented by SEQ. ID. NO:
 1. 2. Themethod for producing inosine according to claim 1, wherein theinactivation is induced by one or more mutation methods selected fromthe group consisting of insertion of one or more base pairs in rih IIgene, deletion of one or more base pairs in the gene, transition ortransversion of base pairs by inserting nonsense codon in the gene. 3.The method for producing inosine according to claim 1, wherein themicroorganism is Corynebacterium ammoniagenes CJIG650 (KCCM-101828P). 4.The method for producing inosine according to claim 1, wherein themicroorganism is further modified to over-express the gene encoding5′-nucleotidase having the nucleotide sequence represented by SEQ. ID.NO:
 2. 5. The method for producing inosine according to claim 5, whereinthe microorganism is Corynebacterium ammoniagenes CJIG651(KCCM-101829P).